The present invention relates to novel urease which is capable of decomposing urea contained in alcoholic beverages and fermentation mashes and causes deterioration of the properties of alcoholic beverages, as well as to a method of producing the same, and relates particularly to a novel form of urease which has an optimum reaction pH value in the acid region and resistance to acidic pH and alcohols and which is produced by a novel microorganism belonging to the genus Arthrobacter, as well as to a method of producing the urease.
Fermented foods and beverages such as Japanese sake, beer, wine, soy source and the like contain urea which is converted from arginine, an amino acid, during fermentation with the aid of arginase which is an enzyme produced by the yeast or molds that take part in the fermentation. Although alcoholic beverages generally contain 2 to 80 ppm of urea, the content of urea in some cases reaches 100 ppm or more when mash is fermented for a long time for the purpose of reducing the lees and increasing the yield.
If an alcoholic beverage contains a large amount of urea, the alcoholic beverage becomes bitter, is discolored and suffers from deterioration of the quality of taste and smell after heat sterilization or long periods of storage.
Ethyl carbamate, a highly carcinogenic compound, is sometimes detected in alcoholic beverages, which is the cause of serious problems [J. Agric. Food Chem., 24, 323 (1976)]. Although the mechanism by which ethyl carbamate is formed has not yet been elucidated, the most plausible hypothesis is that ethyl carbamate is mainly produced from urea or carbamyl phosphate. In other words, it is considered that urea and ethyl alcohol which coexist in alcoholic beverages nonenzymatically react with each other to produce ethyl carbamate.
In order to prevent deterioration of quality, alcoholic beverages are these days required to be sterilized by heating at relatively high temperatures (65.degree. C. or higher) for a certain period (10 minutes or more), and brewages that are stored for a long time as alcoholic beverages have been increasingly used in various fields. Accordingly, a known method of preventing alcoholic beverages from experiencing discoloration and deterioration in flavor due to heating or long-term storage and of preventing the formation of ethyl carbamate is disclosed in Japanese Patent Publication No. 20830/1981. In this method a urease preparation obtained from a certain plant (Jack bean) is added to decompose urea into carbon dioxide and ammonia. In regard to distilled liquors, it is also thought to be effective from the viewpoint of preventing ethylcarbamate formation for the fermented mash that is to be distilled to be first treated with urease for the purpose of decomposing any urea in the mash, because the formation of ethylcarbamate is accelerated at the high temperatures that subsist during distillation.
No urease having suitable characteristics for this purpose is, however, known at present. In other words, it is known that the optimum pH of many kinds of urease for enzyme reactions is generally neutral or weak alkaline (see Japanese Patent Publication No. 55119/1985 and Japanese Patent Public Disclosure Nos. 17987/1984 and 257183/1986), and that these ureases exhibit very little activity and are considerably unstable at an acidic pH or at a high concentration of alcohol as when present in alcoholic liquors [Jack bean urease, Eur. J. Biochem. 73, 185 (1977), Bacillus pasteurri urease, The Enzymes 3rd ed. 4, 1 (1971)]. It is therefore an object of the present invention to provide urease having characteristics that allow the urea to be efficiently decomposed in fermented foods and beverages (particularly alcoholic liquors), that is, to provide a urease having an acidic optimum pH value for reaction and a desirable degree of resistance to acidic pH and alcohol.
Although it is known that Arthrobacter oxydans, which is a bacterium belonging to the same genus as that of the strain of the present invention [Arch. Microbiol., 139, 355 (1984)], produces a urease, it has a neutral optimum pH for reaction and thus fails to meet the objectives of the present invention. This urease is thus the same as many kinds of urease derived from other bacteria. A urease produced by Lactobacillus fermentum, which is an anaerobic bacterium, is known to have an optimum pH value for reaction in an acid region [Appl. Environ. Microbiol., 37, 379 (1979)]. The urease produced by this strain, however, has the disadvantage that the urease strongly binds to the membrane of the bacterium, and thus cannot be easily purified, rendering it unsatisfactory.